Review

Received: 19 April 2016 Revised: 26 September 2016 Accepted article published: 28 October 2016 Published online in Wiley Online Library: 29 November 2016

(wileyonlinelibrary.com) DOI 10.1002/jsfa.8122

Bromelain: from production to

commercialisation
Aizi Nor Mazila Ramli,a* Tuan Norsyalieza Tuan Aznana and Rosli Md Illiasb

Abstract
Bromelain is a mixture of proteolytic enzymes found in pineapple (Ananas comosus) plants. It can be found in several parts of
the pineapple plant, including the stem, fruit, leaves and peel. High demand for bromelain has resulted in gradual increases
in bromelain production. These increases have led to the need for a bromelain production strategy that yields more purified
bromelain at a lower cost and with fewer production steps. Previously, bromelain was purified by conventional centrifugation,
ultrafiltration and lyophilisation. Recently, the development of more modern purification techniques such as gel filtration, ion
exchange chromatography, affinity chromatography, aqueous two-phase extraction and reverse micelle chromatography has
resulted in increased industrial bromelain production worldwide. In addition, recombinant DNA technology has emerged as
an alternative strategy for producing large amounts of ultrapure bromelain. An up-to-date compilation of data regarding the
commercialisation of bromelain in the clinical, pharmaceutical and industrial fields is provided in this review.
© 2016 Society of Chemical Industry

Keywords: pineapple; bromelain; production; commercialisation

INTRODUCTION cosmetic, pharmaceutical and clinical sectors. Applications include

Proteases are hydrolase enzymes that catalyse the hydrolysis of
peptide bonds from the polypeptide chain. This hydrolysis can
take place near the ends of polypeptide chains (exopeptidases) or
within the peptide itself (endopeptidases). Proteases are ubiqui-
tous and have been found in a wide variety of organisms, including
plants, animals and microorganisms. The use of plants as a source
of proteases is governed by the availability of land for cultivation
and the suitability of climatic conditions for growth. The produc-
tion process of plant proteases is time-consuming.1,2 Pineapple
(Ananas comosus) is the most common plant from which proteases
are extracted.3 Ananas comosus is grown in tropical and subtropi-
cal regions across the globe, including China, Indonesia, Malaysia,
Thailand, the Philippines, Kenya and India. Pineapple is also the
second most popular plant in the world owing to its use as both
a food and a medicinal supplement.4,5
Commonly used plant proteases include bromelain, papain, ker-
atinases and ficain. Bromelain is a mixture of proteolytic enzymes
as well as phosphatases, glucosidases, peroxidases, cellulases
and glycoproteins.6 At first, the name ‘bromelain’ was applied
only to the enzyme mixture produced in fruits. Now, ‘bromelain’
is applied to any protease extracted from any member of the
Bromeliaceae family; Bromeliaceae family members usually pro-
duce large amounts of proteases that have no apparent func-
tion in plant growth and development.7 Bromelain is abundant
in several parts of the pineapple plant, including the stem, fruit,
leaves and peel.8,9 Only the stem and fruit produce high amounts
of bromelain, however.10 Bromelain concentrations in pineap-
ple stems are higher than those in the fruit; moreover, pineap-
ple stems are cheap compared with the fruit, which is normally
consumed. According to Muntari et al.,11 stem bromelain is more
widely used in industry than fruit bromelain.12 Bromelain has a
1386
platelet aggregation, fibrinolysis, cancer treatment, modulation of
immune replication, potentiation of antibiotic effects as well as
mucolytic and gastrointestinal actions.6,8,13
In the biotechnology industry, the isolation and purification of
biological products represent approximately 80–90% of total pro-
duction costs. Thus the development of simple, viable methods
of protein purification or the invention of new biotechnological
processes and downstream processing strategies is essential to
ensure the cost-effectiveness and efficiency of product recovery
from crude plant extracts.6 Throughout the bromelain purifica-
tion process, progressive concentration of crude pineapple juice is
required to maintain bromelain activity and to standardise the pro-
teinase composition, as the enzymes tend to be deactivated spon-
taneously. Owing to these limitations, much research has been
performed in attempts to reduce the overall cost of bromelain
production.11,14 Some studies have also been performed to deter-
mine viable bromelain extraction and purification methods.
The purpose of this review is to provide an in-depth sum-
mary of conventional bromelain production strategies and to
discuss the limitations of these strategies. The review will also
provide a thorough description of bromelain production via mod-
ern purification techniques such as gel filtration, ion exchange
∗ Correspondence to: ANM Ramli, Faculty of Industrial Science and Technology,
Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan,
Pahang Darul Makmur, Malaysia. Email: aizinor@ump.edu.my
a Faculty of Industrial Science and Technology, Universiti Malaysia Pahang,
Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang Darul Makmur,
Malaysia
b Department of Bioprocess Engineering, Faculty of Chemical Engineering, Uni-

number of biotechnological applications, especially in the food, versiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

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Bromelain: from production to commercialisation
chromatography, affinity chromatography, aqueous two-phase
extraction and reverse micelle chromatography. A novel strategy
for bromelain production involving recombinant DNA technology
will also be discussed. Finally, a review of the current literature
regarding the commercialisation of bromelain and a discussion
of the future prospects of this enzyme will be provided. It is
hoped that this review paper will offer relevant and useful data
to researchers investigating bromelain or other related enzymes
(e.g. proteases).
PRODUCTION OF BROMELAIN
Plant proteases are commonly produced in the early stages of
fruit development; as fruits mature, these proteases virtually
disappear.15 For example, in figs and papayas, high protease levels
are only found when the fruits are green.16 Compared with other
plants, pineapple is unique in that the fruit contains a relatively
high protease concentration (bromelain) when mature. Pineapple
bromelain is not present in the early stages of fruit development,
but bromelain levels increase rapidly as the plant matures.17 Com-
mercial bromelain is not cheap, with prices approaching US$2400
kg−1 .11 Bromelain production is very expensive, increasing the
market value of bromelain.
To produce highly pure bromelain, several processes must be
performed, including extraction, purification, drying and packing.
The purification process is the most important, as this process
affects the purity and overall processing costs of the end product.14
The extraction of bromelain from pineapple begins with grinding
parts chosen for purification without water in a domestic juicer.
The juice that is extracted consists of a crude enzyme extract.
Generally, bromelain is prepared from cooled pineapple juice
via centrifugation, ultrafiltration and lyophilisation, resulting in a
yellowish powder. Although enzyme yield is a critical parameter
for industrial enzymes, purity is even more important.8,18
To date, no guidelines regarding bromelain purification have
been published. The establishment of such guidelines is a neces-
sary step for laboratory studies.19 Therefore a number of studies
using conventional and modern approaches have been performed
to determine the best procedure for bromelain purification.20 An
overview of these conventional and modern approaches is shown
in Table 1.
Conventional approach
Currently, bromelain is commercially purified using a conventional
approach. Three methods are commonly used to purify bromelain,
involving the following processes: centrifugation, ultrafiltration
and lyophilisation.11 The bromelain purification process begins at
the end of the extraction process. The pineapple juice mixture
must be homogeneous prior to commercial use; homogeneity can
be achieved via several purification processes designed to sepa-
rate components from heterogeneous mixtures. Prior to purifica-
tion, centrifugation is performed to remove solid compounds.21
Centrifugation improves the homogeneity of the bromelain solu-
tion by partially disrupting pineapple cells, releasing intracellular
enzymes without denaturation.22 Gautam et al.23 report that cen-
trifugation of stem bromelain at 448 × g results in a higher enzyme
concentration than fruit bromelain, which requires centrifugation
at 4032 × g to achieve maximum enzyme activity.
Further purification processes are also required, as centrifu-
gation only manages to produce a crude and heterogeneous
bromelain fraction that may contain additional impurities.24,25 In a
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conventional bromelain purification process, ultrafiltration follows
centrifugation. Ultrafiltration is the best method for concentrating
proteins and has been widely used at lab and commercial scales.
Ultrafiltration membranes have molecular weight cut-offs ranging
from 3 to 100 kDa.26 Hebbar et al.27 found that a 5 kDa molecular
weight cut-off membrane can retain bromelain (23–28 kDa) while
separating out lower-molecular-weight solutes. Ultrafiltration
(∼100 min) is typically performed until the volume of the reten-
tate is reduced to 50 mL (i.e. 5-fold reduction). The concentration
of bromelain in the retentate can be increased 8.9-fold. Concen-
trated bromelain is more stable than dilute bromelain and exhibits
virtually no loss of biological activities. Lopes et al.28 report the
use of microfiltration and ultrafiltration (10 kDa) to produce a pure
bromelain extract from A. comosus L. Merr. juice. The authors found
that 85% of bromelain activity can be recovered via microfiltration
(enzyme specific activity of 0.109 units mg−1 ), while 100% of
bromelain activity can be recovered via ultrafiltration. Moreover,
ultrafiltration produced a 10-fold concentrated bromelain extract.
The last purification step in the conventional approach is
lyophilisation. Lyophilisation, or freeze-drying, is used to remove
bound water from the ultrafiltered bromelain.29 Soares et al.30
found that bromelain is susceptible to denaturation after lyophili-
sation, losing as much as 48.6% of its initial activity. This loss of
activity results from interruptions to the hydration layer, which
occur when water around the protein is extracted into a matrix
of ice crystals. Owing to this loss of hydration, the protein ceases
to exist in solution. Sensitive proteins, in particular enzymes, can
become denatured during the transition of water from a solid
to a gas. To minimise denaturation, cryoprotectants can be used
to decrease the tension placed on bromelain by ice crystals.29
Lyophilisation generates a variety of stresses that tend to desta-
bilise or unfold/denature unprotected proteins. Different proteins
tolerate freezing and/or drying stresses differently, such as solute
concentration, formation of ice crystals and pH changes. All of
these stresses can denature proteins to various degrees. Thus
stabilisers are often required in protein formulations to maintain
protein stability during the freezing and drying processes.31 Even
after successful lyophilisation, the long-term storage stability of
some proteins may be very limited, especially at high tempera-
tures. In several cases, solid state protein stability has been shown
to be equal to or lower than liquid state stability, depending
on storage temperature and formulation composition.32 Bresolin
et al.15 lyophilised bromelain after performing precipitation, resus-
pension and desalting. The authors performed lyophilisation to
lessen the moisture content of the sample. However, the lyophili-
sation process reduced the specific activity of bromelain to 5.2%
of its original value. This activity loss is similar to the loss observed
by Devakate et al.9 with fruit bromelain.
Modern approach
Although conventional approaches are the most frequently used
bromelain purification techniques, conventional processes can be
unstable and expensive.33 The fast pace of the modern era requires
that new approaches be developed for bromelain purification.8
Several reports have implied that the preparation of pure brome-
lain is difficult because bromelain is not homogeneous.13,27 If
developed, a viable technique for bromelain purification must be
effective and economically feasible.32 In addition, a viable tech-
nique must maintain the chemical and physical properties of the
original bromelain molecules. Modern techniques such as gel fil-
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tration, ion exchange chromatography, affinity chromatography,
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Table 1. Purification of bromelain using conventional and modern purification approaches
Purification approach Technique involved
Conventional approach • Centrifugation
• Ultrafiltration
• Lyophilisation
Modern approach • Gel filtration
• Ion exchange chromatography
• Affinity chromatography
• Aqueous two-phase (ATP) extraction
• Reverse micelle extraction
aqueous two-phase extraction and extraction via reverse micelles
have been applied to the challenge of bromelain purification.34
Despite their limitations, conventional approaches can still be
used in pre-purification steps to concentrate and increase the
recovery of the target bromelain. Once a suitable concentrate
is obtained, subsequent purification steps to remove remaining
impurities and increase final purity can be performed using mod-
ern approaches.14
Gel filtration
Gel filtration, which functions via separation based on molecular
size, is generally used during the purification of proteins, polysac-
charides, nucleic acids and other biological macromolecules. Gel
filtration partitions molecules between a mobile phase and a sta-
tionary phase comprising a porous matrix with defined porosity. A
study by Suh et al.35 reported that the purification of stem brome-
lain via gel filtration chromatography using CM-Sephadex C-25
and Sephadex G-100 resulted in 45.9-fold concentration and a final
specific activity of 1990 units mg−1 . Purification of fruit brome-
lain using gel filtration chromatography (Sephadex G-100) with a
combination of DEAE-Toyopearl 650C and hydroxyapatite resulted
in 18-fold concentration and a final specific activity of 4900
units mg−1 . It was later discovered that combining ion exchange
with gel filtration chromatography using carboxymethylcellulose
(CMC) and Sephadex G-50 resin can yield pure or homogeneous
bromelain. Costa et al.6 purified bromelain using CMC resin fol-
lowed by Sephadex G-50 resin, producing a purified product with
an activity of 390.75 units mg−1 . The product retained 89% of its
initial activity and was 16.93-fold concentrated. Hernández et al.36
followed a similar purification process but changed the order, first
performing gel filtration and then performing ion exchange chro-
matography. Gel filtration with Sephadex G-100 yielded 87.31%
of the initial proteolytic activity. However, the second purifica-
tion step using CMC decreased bromelain activity to 41.15% of
the initial activity. Compared with Sephadex G-100, purification
with Sephadex G-50 results in higher proteolytic activities. In addi-
tion, using Sephadex G-50 rather than Sephadex G-100 can reduce
costs by 50%. The higher recovery and lower cost of Sephadex G-50
may help make the purification process economically viable.
Ion exchange
Ion exchange chromatography is another approach that has been
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used recently to increase bromelain specific activity. Generally,
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ANM Ramli, TNT Aznan, RM Illias
Advantages Disadvantages
• Increases bromelain recovery • Tedious
in pre-purification steps • Frequently results in low enzyme
yields
• Provides higher selectivity • Expensive for product recovery
and purity of the end product • Stability concerns
• More effective and more eco-
nomical for bromelain purifi-
cation
• Enhances overall enzyme
yields
• Lessens the number of steps
involved in the downstream
processing of bromelain
there are two types of ion exchange chromatography: anion and
cation exchange chromatography.37 Mixed (anion and cation)
columns are commonly used for protein purification, as pro-
teins are complex ampholytes that have both positive and neg-
ative charges. Anion and cation exchange columns both have
resin stationary phases, but the resins contain different functional
groups: anion exchange resins contain positively charged primary
or quaternary amine functional groups, while cation exchange
resins contain negatively charged sulfonic acid groups or car-
boxylic acid groups of negative charges.38 Two weak ion exchange
resins that can be used for protein purification are CMC and
diethylaminoethyl-cellulose (DEAE-cellulose). CMC is negatively
charged, so it is a weak cation exchanger; DEAE-cellulose is posi-
tively charged, so it is a weak anion exchanger. Salts or electrolytes,
which weaken the ionic interactions between proteins and the col-
umn, can be used to elute bound proteins from the column.39
Cysteine proteinase, which is also known as ananain, was pre-
viously isolated from stem bromelain using a combination of
affinity chromatography and cation exchange chromatography.40
Fruit bromelain has also been purified successfully using ace-
tone precipitation, affinity chromatography and cation exchange
chromatography.41 These techniques recovered 85% of the active
cysteine proteinase. Devakate et al.9 report that higher-purity
bromelain extract can be obtained from fruits via cation exchange
chromatography. Using cation exchange chromatography,
Devakate et al.9 obtained a 10-fold concentrated pure brome-
lain product: specific enzyme activity was 590.9 units mg−1
in the eluate and 58.8 units mg−1 in the crude juice. Further-
more, Devakate et al.9 obtained an elution efficiency of 97.6%
and found that the purity of the bromelain obtained via ion
exchange chromatography was 3.3-fold higher than that obtained
via precipitation. As discussed in the previous section, Costa
et al.6 combined CMC cation exchange chromatography with
gel filtration for bromelain purification, producing a 3.01-fold
concentrated bromelain product that retained 93% of its initial
proteolytic activity. Another group of researchers purified stem
and fruit bromelain using anion exchange chromatography with
different concentrations of sodium chloride (NaCl) in Tris-HCl
buffer.23 The use of DEAE-cellulose resin in this study maintained
the structural integrity of the purified bromelain and led to higher
proteolytic activity in the product compared with the crude
extract. From enzymatic assays of ion exchange eluates, it was
found that a maximum amount of NaCl is required for maximum
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Bromelain: from production to commercialisation
TARGET PROTEIN
IS COLLECTED IN CALIBRATION OF
A PURIFIED, REPEAT WITH LIGAND USING
CONCENTRATED ANOTHER SAMPLE BINDING BUFFER
FORM
ELUTION OF
ADDITION OF
MOLECULE
SAMPLES
FROM THE
LIGAND
BINDING OF
MOLECULE TO
THE LIGAND
Figure 1. Affinity chromatography separation procedure.
bromelain proteolytic activity. The use of different resins dur-
ing ion exchange chromatography is important for bromelain
activity.6,9
Affinity chromatography
Using affinity chromatography for protein purification is a good
strategy for eliminating downstream steps, increasing yields and
thereby improving process economics.42 In affinity chromatogra-
phy, the separation of biomolecules is based on highly specific and
reversible biological interactions between two molecules, such as
an enzyme and its substrate. An interacting molecule (affinity lig-
and) is first immobilised onto a solid matrix to create a stationary
phase. The target molecule is then provided in the mobile phase.
The affinity separation procedure is shown in Fig. 1.
Bobb43 describes the purification of bromelain from pineapple
stems via affinity chromatography. The author successfully iso-
lated homogeneous stem bromelain from the acetone powder
of a crude plant extract via a single passage through a column
of agarose-???-aminocaproyl-D-tryptophan methyl ester (ACTME)
coupled to Sepharose 4B. Based on this result, ACTME strongly
adsorbs stem bromelain, as sodium dodecyl sulfate polyacry-
lamide gel electrophoresis (SDS-PAGE) reveals a single band
at 23 kDa. Rowan et al.41 performed a similar study with dif-
ferent types of affinity chromatography columns. The authors
used a Sepharose-Ahx-Phe-GlySc column to separate the major
proteinases comprising stem and fruit bromelain from the het-
erogeneous mixture obtained from the pineapple plant. Fruit
bromelain was successfully purified using an elution step with
mercury(II) chloride (HgCl2 ), resulting in an enzyme recovery
of 90%. Muntari et al.,18 who studied recombinant bromelain,
found that the soluble and insoluble fractions of bromelain can
be successfully purified using affinity chromatography (Ni-NTA
His · Bind resin). To prevent non-specific adsorption, the authors
purified bromelain almost to homogeneity in a single affinity
purification step. The soluble and insoluble bromelain fractions
were concentrated 3.7- and 2.7-fold with yields of 64 and 60.5%
respectively. A higher purification fold and yield were obtained for
the soluble form of bromelain.
Aqueous two-phase extraction
Aqueous two-phase (ATP) extraction systems are widely used
for the separation and purification of biomolecules. Because
the two phases consist of 70–80% water, these systems reduce
enzyme denaturation.44 ATP extraction can remove undesirable
by-products such as unidentified polysaccharides, pigments and
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interfering proteins that lower enzyme activity. In addition, ATP
extraction is low-cost and can be operated continuously. ATP
extraction is environmentally friendly and is an attractive alterna-
tive for the separation and purification of biomolecules.45 How-
ever, ATP extraction is based on mass transfer between two immis-
cible liquid phases. Separation occurs when two polymers (or
one polymer and a salt) are added to the system, resulting in
the partitioning of a specific group of molecules between phases
via the formation of two aqueous immiscible phases. Owing to
the molecular surface characteristics of biomolecules, which make
these molecules susceptible to denaturation in the presence of
organic solvents, a limited number of solvents can be used for
separation.46
ATP extraction is an attractive method for the downstream
processing of proteins in a single unit operation and has been
effectively used for large-scale enzyme separation and purifica-
tion. Purification of a target protein using ATP extraction can
be achieved by controlling a wide range of system parameters.
Sankaran et al.13 found that the speed, simplicity and scalability
of ATP extraction facilitate its use in the large-scale purification
of bromelain. The authors produced an ultrapure bromelain using
an ATP system comprising 8% (w/w) polyethylene glycol (PEG)
and 15% (w/w) ammonium sulfate, obtaining maximum protease
activities of 3958 and 3652 units mg−1 from stem and fruit brome-
lain respectively.
ATP extraction was first used for the separation and purification
of bromelain and polyphenol oxidase (PPO) from pineapples (A.
comosus L. Merr.) in 2008.24 This initial study was performed to
determine the effects of parameters such as molecular weight of
the phase-forming polymer, concentrations of the phase-forming
components and system pH on the partitioning of bromelain
and PPO. Bromelain preferentially partitioned to the top phase,
while PPO preferentially partitioned to the bottom (potassium
phosphate) phase. When the molecular weight of the PEG in the
system was increased, the partition coefficients of both bromelain
and PPO decreased. The phase composition of the system was
found to significantly affect enzyme partitioning and the degree
of purification. This PEG/potassium phosphate system was able
to recover 228% of the initial bromelain activity with a 4.0-fold
purification factor and recover approximately 90% of the initial
PPO activity with a 2.7-fold purification factor.
Ketnawa et al.12 also used ATP to extract bromelain from pineap-
ple peels. The authors tested various PEG polymers to determine
the influence of PEG molecular weight and salt concentration
on bromelain activity, partially confirming the results of a pre-
vious study by Harris.47 Ketnawa et al.12 tested several biphasic
systems containing PEG 1000, 2000 or 3000 kDa with different
salt concentrations. Salts are used in ATP extraction processes to
improve the partitioning of target molecules between phases.
After phase separation, a PEG-rich top phase and a salt-rich bot-
tom phase are obtained. The highest enzyme activity (5.23 units
mg−1 protein) and a yield of 108.45% were obtained with a top
phase consisting of 15% PEG 3000 and 20% MgSO4 in water. In
all of the ATP extraction processes tested, bromelain partitioned
favourably into the polymer phase. The partitioning of enzymes
with hydrophobic characteristics into the polymer phase was
especially favoured. PEG was used by Ketnawa et al.12 because it is
non-toxic and does not harm the active protein. PEG can interact
with cell membranes, however. In PEG/salt systems, the partition-
ing of biomolecules is influenced by the volume exclusion effect
of the polymer in the polymer-rich phase and by salting out in the
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salt-rich phase.
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Reverse micellar extraction
Separation via reverse micellar extraction occurs by protein
solubilisation towards inner micellar cores. To obtain suitable
extraction conditions, the reverse micellar method is some-
times modified. The modified reverse micellar extraction method
can be used for the selective extraction and concentration of
proteins/enzymes. Reverse micellar extraction is a simple and
energy-efficient process.48
Chaurasiya et al.48 found that reverse micellar extraction can
selectively separate bromelain from a crude extract containing
PPO, peroxidase and cellulose. Reverse micellar extraction con-
sists of forward extraction with an organic phase containing
cetyl trimethylammonium bromide (CTAB), iso-octane, 1-hexanol
and 1-butanol and an equal volume of aqueous phase, followed
by back extraction. Back extraction is performed by mixing the
reverse micellar phase obtained from the forward extraction step
with a fresh aqueous phase. Chaurasiya et al.48 obtained a brome-
lain recovery of 85% with a purification fold of 4.0. In addition, the
purified bromelain exhibited better meat tenderisation capabili-
ties than commercial bromelain.
Hebbar et al.49 optimised the reverse micellar extraction condi-
tions for the purification of bromelain from crude pineapple core
extracts. The optimised small-scale (10 mL) conditions for forward
extraction (150 mmol L−1 CTAB, 0.10 mol L−1 NaCl, feed pH 8.0) and
back extraction (acetate buffer pH 4.2, 0.5 mol L−1 KBr) resulted in
an activity recovery of 106% and a purification fold of 5.2. Hebbar
et al.49 also optimised the reverse micellar extraction conditions
for a slightly higher scale (phase volume of 250 mL), finding that
all of the small-scale (10 mL) conditions could be used except for
the duration of phase mixing. At the larger scale, the mixing dura-
tion was increased from 60 to 90 min in the forward and back
extraction steps for efficient phase mixing. Compared with the
small-scale purification, a marginal reduction in recovery (by 10%)
was observed, while purification fold increased from 5.2 to 5.9.
Conventional purification approaches are typically difficult to
scale up, making such approaches unattractive unless the product
is of high value. Reverse micellar extraction is a thermodynamically
stable process that is able to solubilise biomolecules. Moreover,
reverse micellar extraction can be operated continuously and is
simple to scale up.49 Kumar et al.50 describe their optimisation of an
affinity-based reverse micellar extraction and separation (ARMES)
process for the extraction of bromelain from A. comosus L. Merr.
Kumar et al.50 investigated numerous parameters, including con-
canavalin A concentration, counter ligand and surfactant identities
and aqueous phase pH in their study, which ultimately yielded a
purification fold of 12.32 and a recovery of 185.6%. This study is
useful for future scale-up studies. Yin et al.51 studied the scale-up
potential of bromelain extraction via high-speed counter-current
chromatography (HSCCC) coupled with a reverse micelle solvent
system. Yin et al.51 successfully optimised the purification condi-
tions for this coupled system, yielding 123.7 mg of bromelain from
200 mg of crude sample. Subsequent large-scale purification using
the optimised conditions produced 3.01 g of product from 5.00 g
of crude extract.
Novel production strategy of bromelain using recombinant
technologies
Microbial enzymes for enzyme production account for nearly
90% of the total market. Such market domination has spurred
extensive research on recombinant enzymes.18,52 A recombi-
1390
nant enzyme can be defined as an enzyme that is produced
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ANM Ramli, TNT Aznan, RM Illias
intra- or extracellularly via recombinant DNA technology. Recom-
binant enzymes must first undergo purification and formulation
prior to further use.53 With the advancement of molecular genetics
and genetic engineering, it is now possible to clone and express
any gene of interest in a suitable microbial host (e.g. bacteria,
yeasts or fungi), allowing the production of enzymes from other
microorganisms and higher organisms.
From a biotechnological point of view, plant cysteine pro-
teases have been explored inadequately. In the past, plant cys-
teine proteases were primarily used as crude extracts, then in
semi-purified or fully purified forms. Chemical modification of
plant cysteine proteases only began recently.54 The production of
several recombinant cysteine proteases has been reported, includ-
ing papain in Pichia pastoris,11 proteinase IV (glycyl endopepti-
dases) in Escherichia coli and fruit bromelain in transgenic plants.55
The production of stem recombinant bromelain is one of the most
significant recent developments in the field of bromelain research.
Previous reports have described the cloning of the stem bromelain
gene and its expression in E. coli.
The first study involving recombinant bromelain was performed
by Jung et al.55 In this study, the authors cloned and overexpressed
the pineapple fruit bromelain gene (BAA1) in Brassica rapa to
enhance the plant’s defence system against bacterial pathogen.
The study found that the bromelain gene conferred resistance
to soft rot. To date, no one has reported the overexpression of
fruit bromelain in prokaryotes for industrial applications. A study
of recombinant stem bromelain production was performed by
Muntari et al.18 In this study, the authors cloned the stem brome-
lain gene from A. comosus into a pENTR/TEV/D-TOPO vector and
transformed the resulting construct into E. coli BL21-AI competent
cells. After purification, the recombinant bromelain demonstrated
optimal activity at pH 4.6 and 45 ∘ C. Moreover, the recombinant
bromelain exhibited a higher enzymatic activity than commer-
cial bromelain. Further studies on recombinant bromelain were
performed by the same group using a highly purified recombi-
nant bromelain with several advanced features which may not
be adequately present in commercial bromelain.52 In another
study by Nurul and Azura,56 the authors used differential scan-
ning calorimetry (DSC) to measure the thermal and long-term
stability of recombinant bromelain. They found that recombinant
bromelain had enhanced stability at higher temperatures; how-
ever, the stability of the enzyme decreased after 7 days of storage
at 4 ∘ C.
Another study involving the production of recombinant stem
bromelain was performed by George et al.57 The authors initially
cloned the stem bromelain gene into pET32b, expressed the gene
in E. coli BL21 DE3pLysS host cells and purified the resulting
protein using affinity chromatography. George et al.57 found that
the recombinant bromelain had higher activity than the native
enzyme as well as potential bactericidal properties.
COMMERCIALISATION OF BROMELAIN
Bromelain enzymes from different sources may have different pro-
teolytic compositions. Combined bromelain extracts may contain
two or more proteolytic enzymes, increasing their effectiveness.
Although fruit bromelain can be easily extracted from pineapples,
it is not as available commercially as stem bromelain.58 Bromelain
has been the subject of scientific research as a phytomedical com-
pound since 1875.59 The use of bromelain has broadened since
then. At present, bromelain is used clinically, pharmaceutically and
industrially (Table 2).11
J Sci Food Agric 2017; 97: 1386–1395
Bromelain: from production to commercialisation www.soci.org

Immunogenicity
Table 2. Commercialisation of bromelain
Bromelain is used as an adjuvant to stimulate immune responses
Field Commercialisation References during the treatment of chronic inflammatory, malignant and
autoimmune diseases.59 Earlier work on bromelain immuno-
Clinical use Treatment of 59 – 62,65 – 68
genicity was performed by Engwerda et al.,69 who found that
osteoporosis
59,71 – 74
bromelain enhanced IFN-𝛾-mediated nitric oxide and TNF𝛼 pro-
Immunogenicity
59,75,76
duction by macrophages, thereby enhancing the innate immune
Fibrinolysis
59,77 – 79
response against unknown threats.70 In another study, brome-
Diarrhoea
17,80 – 84
lain was found to inhibit T cell signal transduction, blocking
Cancer cells
65,102
the Raf-1/extracellular-regulated kinase (ERK)-2 pathways that
Pharmaceutical use Surgery
8,103 – 105
participate in mitogenesis, apoptosis and cytokine production.71
Potentiation of
Treatment of cells with bromelain was also found to lower CD4+
antibiotics
106 – 110 activation, reducing CD25 expression.72 Increased CD25 levels
Debridement of
burns correlate with the severity of allergic asthma, which commonly
Dermatological 111 – 113 occurs in children.73,74
disorders
Industrial use Tenderisation 91,114 – 117
Fibrinolysis
Baking industry 87,118
Excessive fibrin synthesis, which leads to significant blood clotting,
Textiles 88,90,91
was first reported by Lotz-Winter.75 The application of increased
Tooth whitening 92 – 94
concentrations of bromelain was found to prolong the prothrom-
Cosmetics 97,113
bin inhibition period.59 Prothrombin inhibition results in inactive
prothrombin, reducing the time needed for blood to coagulate.
Bromelain is also a potent fibrinolytic agent that encourages the
Clinical use conversion of plasminogen to plasmin, resulting in improved fibri-
According to the National Institutes of Health (USA), clinical nolysis via fibrin degradation.76
research includes studies which directly involve a certain individ-
ual or group of people or that utilise materials from a human Diarrhoea
source such as observed behaviours or tissue samples gained Studies indicate that bromelain is able to prevent some of the
through direct communication with volunteers who agree to par- effects of the intestinal pathogens Vibrio cholerae and E. coli.77
ticipate in research studies. Bromelain has been used clinically These pathogens produce enterotoxins and cause diarrhoea in
to treat several inflammatory disorders of the musculoskeletal animals.59 To counteract these pathogens, bromelain interacts
system. A number of clinical studies have been performed with with intestinal secretory signalling pathways, which involve sev-
bromelain. eral biomolecule components, including adenosine 3:5-cyclic
monophosphatase, guanosine 3:5-cyclic monophosphatase and
Treatment of osteoarthritis calcium-dependent signalling cascades.78 Some studies sug-
gest a different mechanism of action: active supplementation of
Osteoarthritis is a common musculoskeletal disorder characterised
bromelain has been shown to have antiadhesive effects. These
by pain and disability in joints.60 Based on recent osteoarthri-
antiadhesive effects prevent bacteria from attaching to specific
tis statistics, osteoarthritis in Western countries occurs most fre-
glycoprotein receptors situated on intestinal mucosa by prote-
quently in the USA.61 At 65, osteoarthritis occurs more often in
olytically modifying the receptor attachment sites used during E.
women than in men.62 Several studies have been performed on
coli infection.79
osteoarthritis treatments, and many scientific findings indicate
that bromelain has potential as an osteoarthritis treatment.63,64
Bromelain is thought to be the best natural treatment for inflam- Cancer cells
mation. The first study of bromelain as an anti-inflammatory for Cancerous cells appear as an abnormal growth of cells. Several
rheumatoid arthritis and osteoarthritis was performed in 1964. studies claim that bromelain possesses anticancer properties.80
This initial study was performed to investigate the efficacy of com- According to Chakraborty,81 the anticancer properties of brome-
mon bromelain preparations containing different proteolytic com- lain are primarily applicable to animal (mouse) and human cells.
positions and to investigate the effect of bromelain concentration. Báez et al.82 treated mouse skin tumours with bromelain and
The study found that bromelain was able to reduce inflammation found that bromelain resulted in decreased tumour formation
in osteoarthritis patients.65 and tumour volume as well as apoptotic cell death. Grabowska
Bromelain may also be an alternative to non-steroidal et al.17 applied bromelain F9 and papain on B16F10 to colonies of
anti-inflammatory drugs (NSAIDs). This efficacy of bromelain mouse lung melanoma cells and found that bromelain repressed
was investigated by administering a combination of bromelain, the growth of the melanoma cells in a dose-dependent manner.
trypsin and rutoside to 51 patients. As a control, diclofenac, one Bromelain was also found to reduce the population of GI101A
of the most commonly used NSAIDs, was administered to 52 (breast cancer cell line) cells.83 Finally, a significant reduction in the
other patients.66 Both treatments led to substantial and com- growth of gastric carcinoma Kato III cells was observed after treat-
parable reductions in pain and inflammation within 6 weeks of ment with bromelain.84
consumption.67 However, prolonged use of diclofenac is often
associated with an increased risk of peptic ulcers.68 Daily con- Industrial use
sumption of bromelain was found to reduce the potential risk of Hydrolytic enzymes are widely used industrial enzymes.85 To date,
1391

osteoarthritis compared with NSAIDs.59 proteases remain the most commonly used class of enzymes for

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 www.soci.org
breaking molecular bonds.86 Bromelain, a type of protease, has
been used extensively in the textile, tooth whitening and cosmetic
industries.87
Textile industry
Bromelain improves the dyeing qualities of protein fibres in silk
and wool.88 Silk fibres are produced by cooking silk cocoons to
soften them. During cooking, strong alkaline agents and chemicals
are added that negatively affect the quality of the final silk thread.
Further studies on the use of enzymes during the cocoon cooking
process are important, as enzymes can reduce softening time
while increasing productivity and reducing energy consumption.
A recent research study by Anwar et al.89 reported that fabric
properties were improved when stem bromelain was crosslinked
with 0.25 and 1.25% glutaraldehyde (GTA). Similar results were
reported by Koh et al.90 and Singh et al.91 Koh et al.90 reported that
bromelain can improve the dyeing properties of protein fibres
such as silk and wool. They found that bromelain can eliminate
impurities and scales in wool and silk, improving dye accessibility
while conserving the tensile properties of the fibres. Singh et al.91
found that the addition of a pineapple extract with bromelain
activity with 9.8 mmol L − 1 sodium carbonate at 60 ∘ C during silk
cocoon processing reduced the required softening time from 20 h
to only 30 min.
Tooth whitening
A large number of people seek dentists to improve the appearance
and colour of their teeth. The coupling of intrinsic and extrinsic
colorations on a tooth surface determines the colour of the tooth.
The main causes of extrinsic discoloration are coloured foods, caf-
feinated drinks and smoking.92 According to Chakravarthy and
Acharya,93 dentifrices containing papain and bromelain extracts
exert significant lightening effects compared with control den-
tifrices (Colgate Regular). Discoloured teeth have increased the
demand for tooth whitening products such as toothpaste with
the ability to remove extrinsic stains. Based on data collected by
Kalyana et al.,94 who observed higher lightening levels after brush-
ing compared with control dentifrices, stains can be removed
using papain and bromelain.
Cosmetic industry
Resulting in gentle peeling of the skin, bromelain is used as an
active ingredient in cosmetic products.95 Bromelain can be used
to treat skin complications such as wrinkles, acne and dry skin.
Bromelain works by gently digesting the protein layer of dead cells,
allowing the dead cells to be replaced with younger skin cells.96
FUTURE PROSPECT OF BROMELAIN
One of the most studied proteolytic enzymes is bromelain, which
is typically obtained from pineapple stems and fruits.11 Recent
studies have also found bromelain in other parts of the pineap-
ple, including typical waste components such as the peel, core
and crown.97 The waste from pineapple production has raised
some environmental issues. The outgoing selection and removal
of unwanted processed fruit residues leads to waste accumula-
tion and pollution. Bromelain extracted from pineapple waste
represents a significant valorisation of the wastes generated by the
fruit processing industry.
Many studies have been performed to improve the purification
1392
of bromelain. A few years ago, a bromelain purification study was
wileyonlinelibrary.com/jsfa © 2016 Society of Chemical Industry
ANM Ramli, TNT Aznan, RM Illias
performed involving recombinant bromelain produced in an E.
coli host. Owing to potential endotoxin contamination, this study
raised major concerns regarding user safety. Hence host selection
must be considered carefully. According to Soares et al.,98 fungi
may improve various aspects of the final product. In addition, the
uses of Generally Regarded as Safe (GRAS) organisms for bromelain
production are likely good candidates for products destined for
therapeutic or food applications.
Researchers around the world are now focused on developing
efficient and highly selective catalysts for the synthesis or modi-
fication of complex molecules.99 Various studies have attempted
to produce new active sites on enzymes via mutation of specific
amino acid residues; these novel enzymes eventually possess bet-
ter catalytic activities than the unmodified enzymes. For example,
by covalently modifying a sulfhydryl group, Hilvert and Kaisert100
converted papain into a flavoprotein, a highly effective oxidore-
ductase. This study is a significant breakthrough in the produc-
tion of modified papain enzymes that can catalyse a large vari-
ety of reactions.101 The properties of bromelain may therefore be
improved using protein engineering approaches.
CONCLUSION
Bromelain is of high interest both in industry and in pharmacol-
ogy. Many strategies have been developed to enhance the purity
and activity of bromelain. Although researchers have developed
a number of strategies for the efficient extraction and purifica-
tion of bromelain, some of these approaches require expensive
materials and procedures or are extremely laborious. A number
of modernised approaches, in combination with novel bromelain
production processes, are expected to make bromelain produc-
tion simple, efficient and economical, leading to the development
of cheaper and ultrapure bromelain. Further studies are required to
explore additional strategies for bromelain purification, as well as
protein engineering and recombinant DNA strategies for enhanc-
ing bromelain properties.
ACKNOWLEDGEMENT
Support for this work was provided by Universiti Malaysia Pahang
via research grant UMP RDU 1403169.
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